TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway

Abstract The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.

Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin-embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. deaths occur annually worldwide from cervical carcinoma. 1 In recent decades, the wide implementation of the Pap smear screening program has caused a significant decrease in the incidence of cervical cancer. However, it is still a major public health problem in developing countries. 1,2 Generally, the major therapeutic modality for cervical squamous cell carcinoma (CSCC) (FIGO stage IA2-IIA) is radical hysterectomy and pelvic lymphadenectomy. [2][3][4] The aggressiveness of tumour cells is closely associated with the prognosis of patients with cervical cancer. 2,5 Considerable efforts have been made to elucidate the molecular mechanisms of cell migration and invasion in tumour metastasis and specific methods have been utilized to decrease patient mortality, yet still with limitations. 5,6 Therefore, the identification of tumour-specific markers for the diagnosis of CSCC and the evaluation of its aggressiveness are critical for high-risk patients who require more personalized and urgent clinical intervention.
Tumour suppressor candidate 3 (TUSC3) is encoded by the TUSC3 gene, which is mapped to chromosome 8p22 and contains three prototypical different transcripts. 7 In individual tissues and various stages of embryonic development, the expression of TUSC3 is completely different. [7][8][9][10][11][12][13] More importantly, TUSC3 expression levels correlate with tumour-suppressive or oncogenic function in specific cancer types. Previous studies have revealed that the downregulation of TUSC3 is associated with the incidence of several human cancers, including hepatocellular carcinoma, 10 breast cancer, 11 pancreatic cancer 12 and ovarian cancer. 13 Moreover, it exhibits a positive association with the proliferation, migration and invasion of tumour cells. In contrast to these observations, it was also reported that TUSC3 was overexpressed in several cancer types, such as colon cancer 14 and non-small cell lung cancer. 15 Therefore, it has been suggested that TUSC3 plays intricate and important roles in different types of cancer.
However, the expression pattern of TUSC3 in CSCC and its value for predicting patient survival remain unclear.
In the present study, the expression pattern of TUSC3 was investigated in patients with CSCC. The association of TUSC3 with patient prognosis was analysed, as well as its cellular and molecular function in cervical cancer cell migration and invasion.  All participants had signed written informed consent prior to the investigation. The patients who underwent chemotherapy prior to primary surgical treatment were excluded from this study. All pathological diagnoses of CSCC were confirmed by two independent pathologists. Fresh tumour specimens were timely stored in liquid nitrogen following extraction from the patients. A total of 220 paraffin-embedded specimens were obtained from patients with CSCC and 60 NCTs from benign uterine tumour patients during hysterectomy. ANT (Adjacent Normal Tissue) was normal cervical tissue obtained from cervical cancer patient during radical hysterectomy and obtained cervical cancer tissue from the same patient at the same time. The distance between ANT and paired cancer tissue was equal or >2 cm. Patients were followed up until 31 December 2018. The detailed clinical data are summarized in Table 1.

| Plasmids
The full-length human TUSC3 gene was subcloned into the pSin-EF1α-puro (donation from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences) lentiviral vector using 2019A1515010290); Presidential Foundation of Nanfang Hospital (2018Z014,2017C001, 2018J010); Postdoctoral Science Foundation of Hainan Province, China pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.

K E Y W O R D S
AKT signalling, invasion, prognosis, proliferation, squamous cell cervical carcinoma, TUSC3

| Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from primary tumour tissues and cultured

| Cell growth curve
The cells were plated in 6-well plates (1 × 10 5 cells) and cultured for 6 days. The cells were enzymatic disaggregated and counted every day by using a hemocytometer for the determination of their growth curve. Each cell line experiment was performed in triplicates.

| Immunohistochemistry (IHC)
IHC was performed as previously described. [15][16][17] TUSC3 primary antibody was purchased from Abcam (cat. no. ab230520). TUSC3. An optimal cut-off value was used as follows: a score of ≥6 was used to define tumours with high TUSC3 expression and a score of ≤4 indicated low TUSC3 expression. Positive control staining was performed in human colon cancer tissues and negative control staining was performed without primary antibody.

| EdU assay
The EdU assay was performed as determined by the product specifications (C00031*, Guangzhou RiboBio Co., Ltd).
The cellular layer was wounded using a sterilized tip (1 ml). Following 48 h of cell culture without serum, cell migration was monitored and microscopically photographed. The migratory ability was assessed by measuring the changes in the sizes of the wounded areas of the six fields.

| Transwell assay
The Transwell assay (pore size 8 μm; BD Biosciences) was performed to assess the invasive ability. The membranes of the filters were coated

| Colony formation assay
The cells were resuspended in DMEM supplemented with 10% FBS and plated at a density of 5x10 2 cells/well in 6-well plates.
Following 10 days of cell culture, the colonies were stained with 1% crystal violet (Sigma-Aldrich; Merck KGaA) for 30 sec following fixation with 4% aldehyde for 5 min. The colonies were counted and photographed.

| Tumour xenografts
All experimental procedures were approved by the Institutional Animal

| TUSC3 expression is downregulated in CSCC
The mRNA levels of TUSC3 were downregulated in both CSCC tissues and cell lines, as determined by RT-qPCR ( Figure 1A-C).
Furthermore, the protein levels of TUSC3 were significantly decreased in CSCC tissues (T) compared with normal cervical tissues (NCTs), as evaluated by IHC and Western blotting analyses ( Figure 1D,E).

| TUSC3 regulates CSCC cell proliferation and invasion
To investigate the potential function of TUSC3, stable TUSC3 overexpressing and knockdown Hela and SiHa cells (Hela/SiHa-TUSC3 and Hela/SiHa-shTUSC3) were established (Figure 2A The wound healing assay indicated that Hela and SiHa cells with lower TUSC3 levels exhibited a significantly more extensive wound closure ability compared with that of the control sample ( Figure 3A,B).
The results of the Transwell assay revealed a significant increase in the invasion rate in cells with downregulated TUSC3 levels compared with the negative control group ( Figure 3C,D). Therefore, it was concluded that TUSC3 may play a role in regulating CSCC cell proliferation and invasion in vitro.
We next studied the effect of TUSC3 on CSCC cells and Hela in vivo using the xenotransplantation experiment. Four different numbers (1×10 5 , 1×10 4 , 1×10 3 , 0) of TUSC3 overexpressing (Hela-TUSC3) or knockdown (Hela-shTUSC3) cells mixed with 25% matrigel were collected and subcutaneously inoculated into the inguinal folds of BALB/c nude mice. As shown in Figure 3E,F,G, the tumours formed by Hela-shTUSC3 cells were significantly larger, but the tumours formed by Hela-TUSC3 cells were significantly smaller than the tumours formed by respective control cells. Conjointly, these results demonstrate that knockdown TUSC3 enhanced the tumorigenicity of CSCC cells in vivo.

| Downregulation of TUSC3 levels is associated with CSCC clinical features
The present study explored the expression profile of TUSC3 in 220  We selected statistically significant factors from univariate analysis to include in the multivariate Cox regression analysis. Using multivariate Cox analysis, the data indicated that the downregulation of TUSC3 expression could be used as an independent prognostic factor for PFS (p = 0.005) and OS (p = 0.002) in patients with CSCC (Tables 2 and 3).

| TUSC3 might regulate the AKT signalling pathway in CSCC cells
It was reported in previous studies that the loss of TUSC3 was associated with the activation of the AKT signalling pathway. 20,21 In order to investigate the association between TUSC3 expression and the AKT signalling pathway in CSCC, the phosphoryl- whereas this effect was reversed using SC-79 ( Figure 5C). And

| DISCUSS ION
In the present study, the clinical significance of TUSC3 was assessed in patients with CSCC. The present study revealed that the downregulation of TUSC3 expression increased the proliferation and invasion of CSCC cells, whereas its upregulation exhibited the opposite effects. Furthermore, TUSC3 may modulate the activity of the AKT signalling pathway to promote tumour progression and metastasis.
The downregulation of TUSC3 expression was associated with a high risk of metastasis and unfavourable survival in patients with CSCC. Therefore, TUSC3 may be regarded as a novel prognostic factor for patients with CSCC.
TUSC3 has been characterized as a candidate tumour suppressor gene, which acts as an important signalling hub that modulates a variety of cellular processes. 22 Previous studies have shown that the downregulation of TUSC3 expression is tightly associated with the incidence of several human cancer types, including hepatocellular carcinoma, breast cancer, pancreatic cancer and ovarian cancer, [10][11][12][13] suggesting a functional association between the downregulation of The phosphorylate AKT could result in the overexpression of MMP9. 33 Matrix metalloproteinase (MMP) family is an important protein family associated with the regression of the ECM (extracellular matrix) and EMT. 34,35 Previous studies have shown that silencing of TUSC3-dependent AKT signalling in GBM cells may lead to a high level of proliferation 21 and a similar phenomenon could be observed in prostate cancer. 20 The relationship between AKT signalling pathway and tumour cell progression, migration, invasion in CSCC is also well-known. 36 Therefore, the current study investigated whether the downregu- And the AKT inhibitors MK-2206 could reverse the enhanced CSCC cells proliferation when TUSC3 was knockdown as shown in Figure 5D,E. The AKT activator SC-79 could rescue the inhibited CSCC cells proliferation when TUSC3 was overexpression as shown in Figure 5F. Therefore, knockdown TUSC3 resulted in the

| CON CLUS IONS
The present study demonstrated for the first time that the downregulation of TUSC3 was associated with the enhanced aggressiveness of CSCC cells and poor survival outcomes. This was achieved by using cell functional and survival analysis models. The downregulation of TUSC3 expression promoted CSCC cell metastasis.
TUSC3 might regulate the CSCC cells through AKT signalling pathway. This feature may be used as an independent prognostic factor of CSCC patient survival. Therefore, TUSC3 may become a new prognostic biomarker for patients with CSCC.

ACK N OWLED G EM ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used during the present study are available from the corresponding author upon reasonable request.